Enhanced infectivity of adenovirus type 2 DNA and a DNA-protein complex

174

To determine the role adenovirus 5 early region lb-encoded 21-and 55-kilodalton proteins play in adenovirus productive infection, mutants have been isolated which were engineered to contain small deletions or insertions at 5.8, 7.9, or 9.6 map units. By using an overlap recombination procedure involving H5dl3l4 (A3.7 to 4.6 map units) DNA cleaved at 2.6 map units with ClaI and the adenovirus 5 XhoI-C (O to 15.5 map units) fragment containing the desired mutation, viral mutants were isolated by their ability to produce plaques on KB cell line 18, which constitutively expresses only viral early region lb functions (Babiss et al., J. Virol. 46:454-465, 1983). DNA sequence analysis of the viral mutants isolated (H5dlll8, H5dlllO, H5inl27, and H5dll63) indicates that all of the viruses contain mutations which affect the 55kilodalton protein, whereas d1118 should also produce a truncated form of the 21-kilodalton protein. When analyzed for their replication characteristics in HeLa cells, all of the mutant viruses exhibited extended eclipse periods and effected yields that were reduced to 10% or less of that produced by H5sub309 (parent virus of the^Thutants which is phenotypically identical to wild-type adenovirus 5). When compared with * Corresponding author. MATERIALS AND METHODS Cells and viruses. Monolayer cultures of human KB, 293, (21), HeLa, and KB cell line 18 (a KB cell line that constitutively expresses the bacterial XGPRT gene and the Ad2 Elb gene; 2) cells were grown in Dulbecco modified Eagle medium supplemented with 10% calf serum. Suspension cultures of human KB cells were grown in modified 202

mentioning

confidence: 99%

mentioning

confidence: 99%

Adenovirus type 5 early region 1b gene product is required for efficient shutoff of host protein synthesis

Babiss¹,

Ginsberg²

1984

174

“…Protein-free DNA, purified from adenovirions, can transfect KB cells at low efficiency (5). The DNA-terminal protein complex from such virions is considerably more infectious (3,14). Also, core structures when isolated from the noninfectious virions of mutant Ad2 tsl grown at 39°C are infectious (9).…”

mentioning

confidence: 99%

Adenovirus Type 2 DNA Replicated in Arginine-Starved KB Cells Is Infectious

Auborn

Rouse

1982

“…MATERIALS AND METHODS Cells and viruses. Human cell lines KB and 293 were grown as described previously (2). The 293 cells are a line of human embryo kidney cells transformed by transfection with sheared Ad5 DNA (5).…”

mentioning

confidence: 99%

“…Transfection. DNA transfections were carried out on 293 cells by the "calcium-phosphate-precipitation" method (6) as described by Chinnadurai et al (2). The 293 cells were used for transfection because the infectivity of Ad2 DNA is about 50to 100-fold higher on 293 cells than on KB cells (2, 5).…”

mentioning

confidence: 99%

Physical mapping of a large-plaque mutation of adenovirus type 2

Chinnadurai¹,

Chinnadurai²,

Brusca³

1979

Self Cite

118

We have developed a simple method based on cotransfection of overlapping DNA restriction fragments for construction of recombinants of adenovirus type 2 (Ad2) and Ad5. When Ad2 DNA digested with restriction endonuclease EcoRI was cotransfected with Ad5 DNA digested with SalI, recombination occurred between Ad2 EcoRI-A (map position 0 to 59) and Ad5 SalI-A (map position 45 to 100). Analysis of the recombinant DNAs by digestion with EcoRI or BamHI restriction endonucleases indicated that, as expected, recombination had occurred in overlapping sequences (map position 45 to 59) between the Ad2 EcoRI-A fragment and the Ad5 Sail-A fragment. By using this method, several recombinants were constructed between a large-plaque (Ip) mutant of Ad2 and wild-type Ad5. Cleavage of the recombinant genomes with restriction endonucleases BamHI, EcoRI, and HindIII revealed that the Ip mutation is located within the left 41% of Ad2 genome.